CN115094089A - Establishment method and application of anti-HIV (human immunodeficiency Virus) drug evaluation animal model - Google Patents

Abstract

The invention relates to a method for establishing an anti-HIV virus drug evaluation animal model, which takes an NPG mouse as a construction basis and comprises the following steps: (1) injecting human CBMC cells by tail vein injection; (2) for the mice successfully constructed in the humanized mouse model, infection was performed by intraperitoneal injection of HIV-Nanoluc virus solution. According to the invention, after the human mouse of cord blood CBMC or peripheral blood PBMC is infected by HIV-Nanoluc virus, a large amount of in vivo replication can be carried out in a short period to improve the HIV-1 virus load, so that the effect of evaluating the anti-HIV medicine is more obvious.

Description

Establishment method and application of anti-HIV (human immunodeficiency virus) medicament evaluation animal model

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a method for establishing an anti-HIV (human immunodeficiency virus) medicine evaluation animal model and application thereof.

Background

Acquired Immune Deficiency Syndrome (AIDS), a highly malignant infectious disease with extremely high mortality caused by the Human Immunodeficiency Virus (HIV). The HIV virus invades human body, and can infect human CD 4T cells, destroy the immune system of human body, make patients lose the resistance effect on diseases, and finally cause death. There is currently no effective drug or method for the prevention or cure of this disease by a vaccine. According to the data of the world health organization, nearly 3800 ten thousand AIDS patients exist all the world, and about 150 thousand AIDS patients newly infect AIDS virus in 2020. The number of people infected with AIDS virus in China increases year by year, and the total number of people has broken through millions. With the widespread use of highly active antiretroviral therapy (HAART), the problems of side effects and resistance to HAART have become more serious and new anti-HIV drugs have been developed in large quantities. Aiming at the new medicines, the establishment of an animal model which has high flux, low cost, high efficiency and high speed for screening and evaluating the effectiveness of the anti-HIV medicine is very important. The establishment of an effective HIV infection animal model can provide a basic platform for researches on HIV/AIDS pathogenesis, development of anti-HIV virus medicines, preparation of HIV vaccines and the like.

The HIV infection animal model can be used for metabolic conversion of anti-HIV drugs in human bodies and antiviral effects of the anti-HIV drugs, is a real and objective evaluation on drug effects of the drugs, and plays an important role in research on screening of anti-AIDS drugs. The HIV-1 virus has high host specificity, and can only directly infect a few non-human primate (NHP) animals such as chimpanzees, gibbons and the like except humans. Non-human primates are, of course, good animal models for studying HIV infection, immunity and pathology. Although most non-human primates do not support HIV-1 replication in animals due to restriction by various genetic factors, most non-human primates are susceptible to infection by, for example, Simian Immunodeficiency Virus (SIV) and simian immunodeficiency virus (SHIV), simian aids virus (stHIV) constructed using engineering-based techniques, and SIV and HIV share some similarities in genetic sequence, target cells, and clinical symptoms. Beijing collaborates with AIDS subject group of hospital, has systematically established standardized animal model system of Chinese rhesus monkey AIDS, establishes more than 10 AIDS animal models, and the virus used in these models covers SIV and SHIV strain from different subtype env sources. The standard HIV-1 virus strain HIV-1NL4.3 used in Kunming animal research of Chinese academy of sciences infects northern flattop syndrome, and an HIV-1 infected northern flattop monkey AIDS model is established for the first time.

In addition to non-human primate animal models, non-primate animal models are also commonly used for constructing HIV infection animal models, and the non-primate animal models widely used abroad at present mainly comprise two types of mouse AIDS animal models and cat AIDS (FAIDS) animal models. Feline AIDS (FAIDS) is a feline infectious disease caused by Feline Immunodeficiency Virus (FIV) and is called feline AIDS because it is similar to human AIDS, but human and feline AIDS do not infect each other. The cat AIDS model is mainly applied to researching the dynamics of anti-HIV drugs, drug metabolism and vaccine preparation. Non-primate horses, cattle, etc. have also been the first examples of studies on animal models of AIDS.

Mice are the most commonly used laboratory and clinical laboratory animals for modeling, and mouse aids animal models, such as mouse leukemia virus model, HIV-1 infected chimeric mouse model, and HIV-1 transgenic mouse model, have been successfully constructed. The leukemia virus (MuLV) is injected into a mouse, and the mouse of the model has a series of HIV infection symptoms such as splenic enlargement, lymph node enlargement, immune system function reduction, susceptibility virus and the like, and is widely applied to research and development of anti-HIV medicines, screening and immunoregulation in recent years. The HIV-1 infected chimeric mouse model has 3 main methods: (1) SCID-Hu (Thy/Liv) chimeric mouse model, (2) Hu-PBMCs-NOD/SCID human mouse chimeric model, (3) encephalitis combined with severe immunodeficiency mouse model. The three modeling modes mainly adopt immunodeficient mice or carry out sublethal dose irradiation on host mice to cause the function of an immune system of the mice to be lost, carry out colonization in the bodies of the mice by using human immune cells to construct humanized mice, and then form HIV-infected mouse models through HIV virus infection.

The animal model of non-human primate HIV-1 is considered as an ideal animal model for researching HIV-1 infection, and a plurality of animals such as chimpanzees, gibbons, rhesus monkeys and the like are used for the animal model of HIV-1 infection so far, but a plurality of genetic factors exist in the non-human primates to limit cross-species transmission infection of HIV-1, furthermore, monkey immunodeficiency virus (SIV) and monkey-human immunodeficiency virus (SHIV) and simian AIDS virus (stHIV) constructed based on engineering technology can infect the non-human primates to construct the animal model of AIDS, but the similarity of the SIV and HIV-1 genome sequence is only 45%, and the genetic difference is large, so that SIV can not directly replace HIV-1 for researching HIV/AIDS. The SHIV only embeds partial protein genes of HIV-12.8% -30%, and still has great difference with HIV-1 in gene sequence. Although stHIV-1 and SHIV-vif contain more than 90% of HIV-1 gene sequence, and can replicate in experimental monkeys without AIDS symptoms, the model has not been deeply developed in HIV/AIDS research.

In addition, most of the non-human primates are protective animals in China, and a few of the non-human primates which can be used for animal experiments cannot be massively used for drug screening due to the problems of high price, difficult culture and the like, so that the research on the non-human primate model of the HIV/AIDS disease is difficult. For all of the above reasons, the use of non-human primates for the construction of HIV infected animal models and for large-throughput screening of anti-HIV drugs is not a good choice.

Although animal models of feline aids, animal models of equine aids, and animal models of mouse leukemia virus in non-primate animal models have some clinical symptoms of HIV-1 infection in humans, Feline Immunodeficiency Virus (FIV) has a broader tropism for CD 4T cells, CD 8T cells, or macrophages, but a broader tropism for Ig +/B cells, as does human immunodeficiency virus. Therefore, the constructed animal model of the feline AIDS can not well reflect the dynamics and immunology characteristics of human body infected with HIV, and is not suitable for large-flux screening of anti-HIV drugs in view of higher experimental operation difficulty of the feline. The equine AIDS virus model also has the problems of difficult operation, difficult feeding, high price, long experimental period and the like, and is not suitable for large-flux screening of anti-HIV drugs.

Mice as animal models are increasingly receiving attention from various national scholars, and the murine animals are good model organisms with small size, low cost, simple breeding, convenient operation and rapid propagation, and are widely applied to laboratories and clinical research at present. However, since the mouse itself does not have the human CD4 receptor, it cannot be directly used in the study of HIV-1. The mouse leukemia virus model is one of AIDS virus models, and although the mouse leukemia virus and the HIV-1 belong to the retrovirus family, the similarity of gene sequences is not high, the dynamics and the immunology characteristics of human HIV-1 infection can not be reflected well, and the mouse leukemia virus model is not suitable for large-flux screening of anti-HIV drugs.

HIV-1 transgenic mice can be used for studying infection and replication of partial HIV-1 gene mice, but have a plurality of problems, such as: the problems of different models, accurate positioning and expression of target genes, long-term expression of partial expressed HIV gene products and the like caused by species difference of mice. The concrete points are as follows: (1) the transgenic mouse constructed by HIV-1LTR/Nef/tat, etc. can integrate single or a plurality of gene sequences of LTR/Nef/tat, etc. of HIV-1 into offspring newborn mouse species, and can examine the expression of related genes in a plurality of tissues, but only expresses but does not cause the corresponding diseases of the mouse, does not really realize the infection of HIV-1, and can not be used for screening anti-HIV medicaments (2) HIV full-sequence proviral DNA transgenic mice, although the complete virogene copy is proved to be expressed in partial mice, the expression of the genes and the symptoms of the diseases have great individual difference. The AIDS-like disease phenotype was not observed in the mouse model, and the mouse model had the ability to infect human CD4 positive cells, but not mouse fibroblasts. Meanwhile, because the full-length sequence of the HIV-1 is adopted, the probability of infecting people is extremely high, the operation and culture difficulty of the mouse is increased, and (3) the mouse is a chimeric expression human CD4, CCR5 and CXCR4 transgenic mouse, but the mouse model does not support the replication of the HIV-1 virus and still needs to be prepared by chimeric HIV-1 susceptible human tissues and cells.

Disclosure of Invention

Based on the reasons, the invention provides a method for establishing an anti-HIV virus drug evaluation animal model and application thereof. Specifically, in order to achieve the purpose of the present invention, the following technical solutions are proposed:

the invention relates to a method for establishing an anti-HIV virus drug evaluation animal model, which takes an NPG mouse as a construction basis and comprises the following steps:

(1) hCD45 is detected in mice by injecting human peripheral blood PBMC cells and/or umbilical cord blood CBMC cells through tail vein injection method ⁺ Cell ratio when hCD45 ⁺ The cell proportion reaches more than 30 percent, and the humanized mouse model is judged to be successfully constructed;

(2) aiming at a mouse successfully constructed by a humanized mouse model, injecting HIV-Nanoluc virus liquid into the abdominal cavity for infection, imaging and detecting the proliferation condition of the virus in vivo every week after infection, and when the ROI value of a fluorescence signal reaches 10 ⁶ ～10 ⁷ phototons/s are used for successfully modeling an anti-HIV virus medicament evaluation animal model. According to the invention, after HIV-Nanoluc virus is adopted to infect peripheral blood PBMC or umbilical cord blood CBMC humanized mice, a large amount of in vivo replication can be carried out in a short period to improve the HIV-1 virus load, so that the effect of evaluating the anti-HIV medicine is more obvious.

In a preferred embodiment of the present invention, the extraction of the peripheral blood PBMC cells of human origin or CBMC cells of umbilical cord blood comprises the steps of:

collecting peripheral blood of a healthy person, uniformly mixing the peripheral blood with heparin anticoagulant, performing centrifugal separation to collect upper plasma for later use, mixing the rest blood components with PBS preheated to room temperature in a volume ratio of 1:0.5-2, flatly paving the mixture on Ficoll lymphocyte separation liquid to form a clear separation interface, centrifuging, sucking a middle leucocyte layer into a clean centrifugal tube, adding PBS for cleaning, centrifuging, discarding supernatant, and cleaning for 1-3 times. By adopting the method, the number of the red blood cells can be controlled to be below 5 percent; preferably 2% or less.

In a preferred embodiment of the present invention, the caudal vein is injected with human CBMC cells of 4-6X 10 ⁶ cell/cell.

In a preferred embodiment of the present invention, the injection amount of the HIV-Nanoluc virus solution is 5 to 10. mu.g/vial. By controlling the injection amount of the virus solution within the preferred range, on the one hand, death of the mouse due to injection of the virus solution can be avoided, and on the other hand, successful molding can be achieved within 3 weeks.

Another aspect of the invention relates to the use of the above animal model for screening anti-HIV drugs.

According to the application, the anti-HIV drug is VRC01-CAR-CD3 ⁺ T cell preparation, the VRC01-CAR-CD3 ⁺ The T cell preparation VRC01-CAR lentivirus transduced CD3 positive cells to obtain, the VRC01-CAR lentivirus consisted of signal peptide SP1, anti-HIV gp120 antigen specific single chain antibody VRC01, tag signal Strep tag II, linker peptide, CD8 hinge region, CD28 transmembrane region, 4-1BB co-stimulatory domain and CD3 zeta intracellular signaling domain.

The invention has at least the following beneficial effects:

1. the invention adopts the highly immune-deficient NPG mouse, which can not cause graft antibody host disease (GvHD) and can prolong the HIV infection window period;

2. the invention optimizes the method for extracting PBMC from peripheral blood and CBMC from umbilical cord blood, and has lower red blood cell content compared with PBMC and CBMC obtained by the prior method;

3, the invention compares PBMC and CBMC humanization modeling, and detects and finds that the expression of HLA-DR and HLA-DQ genes directly related to GvHD is 200 times lower than that of CBMC cells, thus proving that the GvHD generation severity of a CBMC humanization mouse is smaller than that of a PBMC modeling mouse, thus striving for a longer window period for in vivo antiviral experiments, and then the humanization mouse models are all modeled by CBMC.

4. The humanized CBMC mouse constructed by the invention can stably establish the human immune ecological environment in a mouse body and further establish a stable HIV-1 infection and virus replication model of a murine animal.

5. The invention carries out gene modification on the basis of NL4-EGFP, replaces EGFP label with Nanoluc label, successfully obtains newly constructed HIV-Nanoluc virus, and cooperates with the prior HIV-Nanoluc virus

The substrate luminescent system catalyzed by Luciferase can intuitively express the effect of HIV-1 infecting humanized mice.

Drawings

FIG. 1: genome Structure design of HIV-Nanoluc (NL4-3) carrying the luciferase Nanoluc.

FIG. 2: and (3) an agarose gel electrophoresis result of the HIV-Nanoluc construction.

FIG. 3: plasmid backbone NL4-EGFP plasmid map and the successfully constructed HIV-Nanoluc plasmid map.

FIG. 4 is a schematic view of: VRC01-CAR structure and its plasmid map for efficacy evaluation.

FIG. 5 is a schematic view of: physical titer assay and infectivity assay for packaged HIV-Nanoluc virus.

FIG. 6: flow cytometry compares the differentiation results of human T cells in vivo in humanized mice constructed by tail vein injection and intraperitoneal injection, as well as PBMC and CBMC.

FIG. 7: HLA-DR, HLA-DQ expression analysis in PBMC and CBMC

FIG. 8: the cookie scoring system assesses GvHD severity 50 and 150 days after humanization modeling PBMC and CBMC.

FIG. 9: tail vein injection CBMC modeling was evaluated by in vivo immune reconstitution of mice 54 days later.

FIG. 10: tail vein injection CBMC modeling mice were evaluated for in vivo immune reconstitution 145 days later.

FIG. 11: flow cytometry detection CBMC humanization modeled differentiation of human T cells in peripheral blood of 4-week mice.

FIG. 12: imaging kinetics of HIV-Nanoluc virus infection of humanized mice and analysis of viral gene expression.

FIG. 13: VRC01-CAR-CD3 ⁺ T CAR-T cell growth curve.

FIG. 14: flow cytometry detection of VRC01-CAR-CD3 ⁺ T cell transduction efficiency analysis.

FIG. 15 is a schematic view of: the invention is used for constructing a humanized HIV animal model and carrying out the efficacy evaluation of CAR-T cells by using a small animal imaging technology.

FIG. 16: QPCR Gene expression levels analysis the therapeutic effect of CAR-T on infected mice.

Detailed Description

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless otherwise specified, the reagents involved in the examples of the present invention are commercially available products, and all of them are commercially available.

Example 1:

1 construction of HIV-infected clones harboring the novel luciferase NanoLuc

In order to facilitate evaluation of HIV infection level in mice, the present invention constructed and verified a recombinant HIV virus HIV-Nanoluc (NL4-3) into which a novel luciferase (Nanoluc) gene was inserted. The construction of the NL4-3 virus plasmid is specifically as follows: firstly, a plasmid containing Nanoluc is synthesized, a Nanoluc fragment (primers are shown in the following table 1) is amplified through a PCR technology, BamHI and Not I enzyme cutting sites are added, and the Nanoluc fragment is recovered through running glue. The HIV-EGFP (NL4-EGFP) plasmid is double digested with BamH I and Not I to obtain the HIV-NL4 plasmid vector backbone and recovered by running gel. And connecting the Nanoluc fragment with the HIV-NL4 plasmid skeleton overnight by using T4-DNA ligase, and verifying the correctness by sequencing, thereby indicating that the recombinant HIV virus HIV-Nanoluc is obtained. The structure is shown in figure 1, the agarose gel electrophoresis result of the Nanoluc fragment amplified by the PCR technology and the agarose gel electrophoresis result of the HIV-EGFP plasmid after double enzyme digestion are shown in figure 2, and the plasmid skeleton NL4-EGFP plasmid map and the successfully constructed HIV-Nanoluc plasmid map are shown in figure 3.

Table 1: primer sequence for amplifying Nanoluc fragment

To facilitate the drug evaluation effect of the subsequent HIV-infected mouse model, the inventors of the present application have constructed a CAR structure using an HIV broad-spectrum neutralizing antibody scFv as an extracellular recognition region, the CAR sequence consisting of a signal peptide SP1, an anti-HIV gp120 antigen-specific single-chain antibody VRC01, a tag signal Strep tag II, a linker peptide, a CD8 hinge region, a CD28 transmembrane region, a 4-1BB co-stimulatory domain, and a CD3 ζ intracellular signaling domain (as shown in fig. 4), and verified the effect of the CAR-T in treating HIV.

NanoLuc luciferase (Nluc) is a small molecule enzyme (19.1kDa) engineered to produce a high intensity, glow-type luminescent signal using a novel substrate, Furimazine. The bioluminescence reaction is independent of ATP, the self-luminous background is low, the light signal is brighter and can reach 10 ¹⁰ And, at the same time, suppressing background luminescence to obtain high detection sensitivity, has been widely used for live animal imaging at present. After viral infection, live animal imaging can be used to quantify and localize viral replication in vivo and to evaluate the antiviral efficacy of the drug. However, the inventors of the present application searched for how to construct NanoLuc and HIV into the same plasmid to ensure that NanoLuc can generate a strong optical signal without affecting HIV infection of model mice, thereby obtaining an HIV-NanoLuc plasmid having a specific sequence according to the present invention. Wherein, the nucleotide sequence of the 5' LTR is SEQ ID NO. 1; the nucleotide sequence of HIV-1 psi is SEQ ID NO. 2; the nucleotide sequence of Gag-Pol is SEQ ID NO. 3; the nucleotide sequence of Vif + Vpr is SEQ ID NO. 4; the nucleotide sequence of Tat + Vpu is SEQ ID NO. 5; the nucleotide sequence of Env is SEQ ID NO. 6; the nucleotide sequence of the Nano-Luc is SEQ ID NO. 7; the nucleotide sequence of IRES is SEQ ID NO. 8; the nucleotide sequence of the 3' LTR is SEQ ID NO. 9.

2 determination of physical Titers after HIV-Nanoluc Lentiviral packaging concentration and verification of the ability to infect PBMCs

Co-transfecting 293T with HIV-Nanoluc plasmid and PEI, collecting culture supernatant after 3 days, centrifuging, purifying and concentrating to obtain HIV-Nanoluc virus, detecting the physical titer of the HIV-Nanoluc virus by using a P24 Elisa kit, and calculating the physical titer of the HIV-Nanoluc virus according to a standard curve of 5-A P24 according to experimental results shown in figures 5-B to 5-D. The NL4-Nanoluc virus after concentration was subjected to infection experiments to verify the virus infectivity. The addition amount of NL4-Nanoluc virus is calculated according to the physical titer of P24, the infected target cells are PBMC activated by PHA, and the expression amount of Nanoluciferase reporter gene is detected by an imager, so that the infection capacity of the virus is reflected. The results of the experiments are shown in FIGS. 5-C and 5-D, and show that NL4-Nanoluc virus emits a fluorescent signal in the reporter gene after infecting PBMC, indicating that NL4-Nanoluc virus has the ability to infect PBMC; meanwhile, the NL4-Nanoluc virus has higher domain fluorescence intensity (indicated by "ROI") under the condition of adding 100pg than the ROI added with 10pg and 1pg, and the ROI is about 10 times of that added with 10pg when 100pg is added, which shows that the NL4-Nanoluc virus packaged in the experiment has better dose dependence.

3 separation and technical optimization of healthy human peripheral blood PBMC and healthy human cord blood CBMC

Collecting peripheral blood of a healthy person, gently mixing the peripheral blood with heparin anticoagulant, subpackaging the peripheral blood into centrifuge tubes for weight balancing, adjusting parameters by using a horizontal rotor centrifuge to ensure that the acceleration and the deceleration are all 1 grade, centrifuging the peripheral blood at room temperature of 700 Xg for 10 minutes, and discarding the upper plasma. Mixing the rest blood components with PBS preheated to room temperature at a ratio of 1:1, spreading on the separating solution of Ficoll lymphocyte to form a clear separation interface, adjusting parameters by using a horizontal rotor centrifuge to ensure that the acceleration and deceleration are all 1 gear, and centrifuging at room temperature of 700 Xg for 25 minutes. And (3) sucking the middle leucocyte layer into a clean centrifugal tube, adding PBS (phosphate buffer solution) for cleaning, centrifuging by 450g, discarding supernatant, and cleaning twice. If the extracted PBMC contain a relatively large amount of erythrocytes, lysis can be performed by adding an erythrocyte lysate. PBMCs obtained by the method are substantially free of erythrocytes and take relatively little time. The extraction method of CBMC in umbilical cord blood is the same, and only the centrifugal force size of 700 Xg is changed into 650 g. The extracted cells were observed under a microscope and no red blood cells were observed.

4 respectively injecting PBMC and CBMC into NPG mice by intraperitoneal injection and tail vein injection, and comparing the humanized modeling of the mice

After 3 days of adaptive culture in 4-week-old NPG mice, the PBMCs and CBMCs isolated from the previous step were injected in vivo for modeling of humanized mice. Two injection modes commonly used for mouse humanization modeling are tail vein injection and intraperitoneal injection at present, wherein the intraperitoneal injection difficulty is low and is adopted by most people. The experiment simultaneously adopts the tail vein injection and the intraperitoneal injection to carry out humanized modeling on PBMC and CBMC injected into the NPG mouse body, and the influence of the two injection modes and two human source cells on the modeling is explored.

The specific experimental mode is that the NPG mice with the age of 5 weeks are selected and divided into 4 groups, and each group comprises 4 mice, so that the weight, the state and the like of the four groups are kept relatively consistent. One group adopts tail vein injection PBMC 5X 10 ⁶ cell/cell, two groups injected intraperitoneally with PBMC 5X 10 ⁶ Three groups of cells/cell adopt tail vein injection CBMC 5 × 10 ⁶ cell/cell, four groups using intraperitoneal CBMC 5X 10 injection ⁶ cell/cell. Observing and culturing in animal house for 3 weeks, collecting peripheral blood of four groups of mice (about 150ul each) by submaxillary venous blood collection method, lysing peripheral blood of mice with erythrocyte lysate special for mice to obtain peripheral blood lymphocytes of mice, counting, and collecting 1 × 10 ⁶ The cell is subjected to flow-type staining of HuCD3/CD4/CD8 flow-type antibody, after being cleaned, the cell is subjected to flow-type on-machine, and the influence of two injection modes and two human source cells on the modeling of the humanized mouse is judged according to the typing result of the human T cells in the peripheral blood of the modeled mouse.

The experimental results are shown in fig. 6, and it is known that in peripheral blood of humanized mice constructed by two human source cells PBMC and CBMC, the content of Hu-CD 4T cells in the caudal vein injection group is higher than that in the intraperitoneal injection group, and the content of Hu-CD 8T cells in the intraperitoneal injection group is higher. Comparing PBMC mice and CBMC mice injected by tail vein at the same time, it can be seen that the content of Hu-CD 4T cells in the CBMC mice is higher. Because HIV mainly infects Hu-CD 4T cells, the experimental result shows that the humanized mouse constructed by the tail vein injection CBMC method is more in line with the requirement of constructing an infection model mouse.

Physiological status analysis and GVHD severity assessment after NPG mouse humanization modeling by 5-tailed intravenous injection of human PBMC and human CBMC

In order to further detect the influence of tail vein injection of human PBMC and CBMC on the survival state of the humanized mice, the peripheral blood of the mice is collected 4 weeks after the NPG mouse is successfully humanized and modeled, and the expression of HLA-DR and HLA-DQ genes directly related to GvHD is detected by flow cytometry. T cells in human PBMC proliferate when stimulated by mouse xenoantigen, while other cell types maintain low levels or die prematurely. The human source cells in the PBMC humanized mouse are mainly T cells, and are suitable for various researches requiring T cell immune response. However, T cells also cause excessive immune attack on recipient mice, which in turn causes graft versus host disease (GvHD). The results are shown in FIG. 7, the PBMC humanized mice highly express HLA-DQ and HLA-DR 200 times higher than CBMC humanized mice, and therefore it can be assumed that GvHD development in CBMC humanized mice is delayed compared to PBMC modeled mice, which can strive for a longer window period for in vivo antiviral experiments.

PBMC humanized mouse CBMC humanized mouse body weight, morphology, behavior, etc. were observed and photographed at 50 days and 150 days simultaneously for the humanization modeling. Meanwhile, mice were evaluated for GVHD according to the general GVHD scoring standard-Cooke scoring system (see Table 2). As shown in fig. 8, when the model was humanized for 150 days, the PBMC humanized mouse began to show symptoms such as hunched and shrug, and had symptoms of GVHD such as lassitude, anorexia, lusterless hair, alopecia, unstable gait, and diarrhea; in contrast, the CBMC humanized mice were in good condition, had little or no hunch, shrug, and depilation, and had high mobility. The GVHD scores of the PBMC humanized mouse and the CBMC humanized mouse are not greatly different after 50 days of humanized modeling, which indicates that the highly immunodeficiency NPG mouse still keeps a better survival state 50 days after humanized modeling; at 150 days of humanization modeling, the GVHD score of PBMC humanized mice was close to 5-fold that of CBMC humanized mice, with significant differences, indicating that PBMC humanized mice develop high-grade GVHD.

Based on experiment result 6, experiment result 7 and experiment result 8, humanized mouse constructed by injecting CBMC through tail vein expresses human CD3 in peripheral blood of mouse ⁺ CD4 ⁺ T cells can be maintained for 150 days without high GVHD, and the window period of the experiment is greatly prolonged, so that the humanized mouse modeling mode adopted in the subsequent experiments is that CBMC is injected through tail vein.

TABLE 2 Cooke Scoring System

Typing detection and evaluation of T cells in mice after 6-tailed intravenous injection of human CBMC (CBMC)

In order to further detect the typing of different tissues of T cells in mice after the tail vein injection of human CBMC, the modeling mice are respectively taken at 54 days and 145 days after the injection for dissection, the peripheral blood tissues, the spleen tissues and the bone marrow of the mice are taken for treatment to obtain blood lymphocytes, the spleen cells and the bone marrow cells, the blood lymphocytes, the spleen cells and the bone marrow cells are counted and taken at 1 × 10 ⁶ The cell is subjected to flow staining of hCD3/CD4/CD8 flow antibody, and after being cleaned, the cell is subjected to flow type computer, and the typing condition of the human CBMC in the NPG mouse is detected according to the T cell typing result of cells at the corresponding tissue part of the modeled mouse.

As shown in FIG. 9, it was found that the NPG humanized mouse constructed by tail vein injection of human CBMC exhibited good humanization effect, as a result of differentiation of hu-CD 4T cells and hu-CD 8T cells in the bone marrow cells, and of differentiation of hu-CD 4T cells and hu-CD 8T cells in the spleen cells, at about 30%, on day 54. It was concluded that human CBMC appeared to differentiate in mouse bone marrow and spleen at 54 days of human CBMC injection, especially in spleen tissue.

Experimental results As shown in FIG. 10, hCD3/CD4/CD 8T cells remained in bone marrow, spleen and peripheral blood at day 154 in NPG humanized mice constructed by tail vein injection of human CBMC. From the results, it can be known that the NPG humanized mouse constructed by the experimental method has a long humanized effect, prolongs the HIV infection time, and can be used for a long experimental period.

Typing detection and evaluation of T cells in mouse peripheral blood after 7-tailed intravenous injection of human CBMC (CBMC)

Purchasing 4 weeks old NPG mouse, adaptively culturing for one week, taking healthy NPG mouse, and injecting human CBMC 5 × 10 into tail vein ⁶ cell/mouse, after observing for 2 weeks, submandibular venous blood sampling detects the proportion of hCD45/CD3/CD4/CD 8T cells in the mouse, and detection is carried out once a week.

Specifically, about 150ul of peripheral blood of all modeled mice is taken by adopting a submaxillary venous blood sampling method, the peripheral blood of the mice is cracked by using special erythrocyte lysate of the mice to obtain peripheral blood lymphocytes of the mice, 1 x 10^6 cells are counted and subjected to flow-type dyeing of hCD45/CD3/CD4/CD8 flow-type antibodies, the flow-type machine is carried out after cleaning, and the construction condition of the NPG mouse humanized modeling is judged according to the differentiation condition of human T cells in the peripheral blood of the modeled mice.

The experimental results are shown in fig. 11, the NPG mice injected with human CBMC for 4 weeks contained more than 40% hCD45+ T cells in the peripheral blood lymphocytes of the mice, and had higher CD3+ CD4+ T cells, which met our infection experimental requirements, indicating that the humanization modeling was successful.

Animal weights were weighed 1 time per week and the humanised modelled and unmodeled control mice maintained steady state body weights. And (3) clinical observation: observations include, but are not limited to, behavioral activity, skin, quilt hair, eyes, ears, nose, abdomen, external genitalia, anus, limbs, feet, respiration. Observations indicate that the humanized modeled mice and the unmodeled control mice are about the same.

Construction of HIV-infected mouse model and detection Standard

Detecting hCD45 in peripheral blood in the last step ⁺ Mice with over 40 percent of T cells are injected with HIV-Nanoluc virus liquid (6 mu g/mouse) intraperitoneally to construct an HIV infection mouse model, imaging is carried out every week after infection to detect the proliferation condition of the virus in vivo, and when the ROI value of a fluorescence signal reaches 10 ⁶ ～10 ⁷ Photons/s were successfully constructed for the infection model.

In the mouse imaging experiment, a Nano-Glo Luciferase experiment system is used, imaging substrates are configured according to the requirements of a specification, 150 mu l of imaging substrates are injected into each mouse through an abdominal cavity, then the mouse is anesthetized through an animal anesthesia system by using isoflurane, then the imaging operation is carried out by using a small animal imaging system, the exposure time is adjusted to 60s, and the other parameters are defaulted.

As shown in FIG. 12-A, the imaging of the abdominal cavity of the humanized mouse 3 weeks after HIV-Nanoluc virus infection showed a high fluorescence signal indicating high titer HIV virus replication. FIG. 12-B is a quantitative result of the fluorescence intensity analysis of FIG. 12-A.

To demonstrate the kinetics of HIV infection in mice, mice were dissected 7 days after HIV-Nanoluc virus infection and 14 days after HIV-Nanoluc virus infection, ground to obtain mouse spleen cells, and counted to 1X 10 ⁶ cell, using RNA extraction kit to extract RNA, measuring concentration after RNA extraction is completed, taking the same mu g of spleen cell RNA to form cDNA by using reverse transcription DNA kit of semer fly, taking 1 × 10 spleen cells ⁶ The cell extracts DNA by using DNAzol, and the concentration is measured after the extraction is finished. The spleen cells were tested for infection kinetics with NL4-1RNA and DNA copy number in spleen cells of an infected mouse model by QPCR experiments using the same μ g of spleen cell cDNA and DNA. The primers and probes used for quantification were gag primers and probes as in table 3.

TABLE 3.1 Fluorogenic quantitative PCR primers and probes

TABLE 3.2 fluorescent quantitative PCR reaction System

TABLE 3.3 fluorescent quantitative PCR reaction conditions

The results of the experiments are shown in FIGS. 12-C, 12-D and 12-E, and with the increase of infection time, HIV-1RNA, DNA copy number and integrated DNA copy number were continuously increased in mice at 14 days after HIV-Nanoluc virus infection, and HIV-1RNA copy number, HIV-1DNA copy number and HIV-1DNA integrated copy number were increased by about 7 times, about 6.5 times and about 15 times respectively 7 days after infection. And the imaging result has higher consistency, so that the effect of evaluating the anti-HIV drug is better.

9VRC01-CAR-CD3 ⁺ Preparation of T cell preparation and determination of transformation efficiency

9.1CAR-CD3 ⁺ T cell preparation

Collecting 60mL of umbilical cord blood of a healthy donor, and separating mononuclear cells by using a Ficoll lymphocyte separation solution, wherein the specific method is the same as the method for separating peripheral blood PBMC. After counting, the appropriate amount of CD3 was used ⁺ Sorting CD3 positive cells by a T cell positive selection magnetic bead kit, and sorting the positive cells by 1.0-2.0 multiplied by 10 ⁶ The cells were cultured in a complete T cell culture medium at a density of 10 cells/mL ⁶ The cells were activated by adding 25. mu.l of human CD3/CD28 activated magnetic beads to the cells.

After 24 hours, adding VRC01-CAR lentivirus according to MOI-3 for transduction, simultaneously adding proper polybrene for mixing uniformly, and placing in CO ₂ And (5) incubating in the incubator, and supplementing a proper amount of complete T cell culture medium after 4 hours to perform liquid change culture.

VRC01-CAR lentivirus was transduced for 24 hours to yield VRC01-CAR-CD3 ⁺ Replacing T cells with fresh complete T cell culture solution, and adjusting viable cell density to 1.0-2.0 × 10 ⁶ mL, continuously culturing and amplifying for 10-20 days, observing and counting every day, performing fluid infusion and amplification culture according to the counted cell number, and always keeping the cell culture density at 1.0-2.0 × 10 ⁶ and/mL. Counting every day to obtain VRC01-CAR-CD3 ⁺ The growth curve of T is shown in FIG. 13.

Collection of VRC01-CAR-CD3 based on predicted cell usage ⁺ T cells, resuspended at 2%Transferring human albumin in 100mL physiological saline into cell reinfusion bag, and heat sealing to obtain VRC01-CAR-CD3 ⁺ And (5) preparing a finished product of the T cell preparation. Or frozen in DMSO containing 10% FBS and the cells were cryopreserved using a programmed cryo-meter.

9.2CAR-CD3 ⁺ T cell transduction efficiency assay

Take 1.0X 10 ⁶ Individual CAR-CD3 ⁺ T cells, incubated with FITC-Strep II for 30 minutes at room temperature, washed twice with PBS containing 2% FBS, and then FITC fluorescence signal was detected by flow cytometry to measure the FITC-positive cell ratio, reflecting the ratio of CAR-positive cells in total cells. VRC01-CAR-CD3 ⁺ T cell transduction efficiency, the results of which are shown in FIG. 14. FIG. 14 shows the successful preparation of VRC01-CAR-CD3 ⁺ The expression rate of the CAR molecules of the T cells and the anti-gp120 reaches 39.6 percent, and the requirements of the animal experiment design of the project can be fully met.

10 in vivo efficacy experiment of CAR-T cells using HIV-infected mouse model

CAR-T treatment HIV infection model drug effect experiment: the ROI value of a fluorescence signal reaches 10 after HIV infection for about one week ⁶ ～10 ⁷ Infection of Photons/s mouse model, frozen VRC01-CAR-CD3 cultured as described above was injected ⁺ T cell 1-5X 10 ⁷ cells/only. Weekly imaging for mouse viral load. And dissecting a mouse at an experimental end point, extracting DNA from spleen tissues and brain tissues of the mouse, performing reverse transcription on the DNA and the RNA to form cDNA, performing QPCR experiment, and determining the virus load of the tissues of the mouse, wherein the experimental result is shown in figure 11.

As can be seen from FIG. 15, the fluorescence signal intensity of the placebo-treated mice increased continuously with time, and VRC01-CAR-CD3 ⁺ T treatment group mice continued to decrease in fluorescence signal intensity with increasing treatment time and remained undetectable in the instrument for two weeks, indicating that CAR-T gradually killed HIV-Nanoluc infected cells in vivo. This revealed that the infection model can be used as a model for evaluating the efficacy of mice.

At 22 days after CAR-T treatment, the placebo and CAR-T treated groups were dissected and spleen cells were harvested at 5X 10 ⁶ Extracting RNA from cells, measuring the concentration after the RNA extraction is finished, taking spleen cell RNA with the same amount, forming cDNA by using a reverse transcription DNA kit, and performing a QPCR (quantitative polymerase chain reaction) experiment to determine the HIV-1RNA copy number of mouse tissues. Another spleen cell of mouse was taken at 5X 10 ⁶ Extracting DNA from cells, extracting DNA by using DNAzol, and determining the copy number and integrated copy number result of HIV-1DNA in spleen tissues by using a QPCR (quantitative polymerase chain reaction) experiment. Taking the same weight of brain tissue to extract DNA and RNA, wherein the RNA is reversely transcribed into cDNA by using the kit, taking the same microgram of DNA and cDNA to carry out QPCR (quantitative polymerase chain reaction) experiment, and determining the copy number and integrated copy number results of the HIV-1DNA in the brain tissue

The experimental results are shown in FIG. 16, and the spleen tissue and brain tissue of the CAR-T cell treated mice and the control mice have significant differences in HIV-1RNA, DNA and integrated DNA copy number. In spleen tissue, the CAR-T cell treated mice had an approximately 3.7-fold reduction in HIV-1RNA copy number, an approximately 3.2-fold reduction in HIV-1DNA copy number, and an approximately 3.7-fold reduction in integrated HIV-1DNA copy number compared to control mice. In brain tissue, the CAR-T cell treated mice had an approximately 5.6 fold reduction in HIV-1RNA copy number, an approximately 4.8 fold reduction in HIV-1 total DNA copy number, and an approximately 2.2 fold reduction in integrated HIV-1DNA copy number compared to control mice. At the same time, the results show that the HIV-1 copy number of the spleen tissues of the mice is far larger than that of the brain tissues. As can be seen from fig. 12, as the humanized mice were infected with HIV virus, the HIV virus load in the mouse spleen became higher.

According to the experimental result of imaging of the small animal and the QPCR experimental result, the infected animal model can be used for evaluating the drug effect of the HIV drug.

The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.

Sequence listing

<110> Wuhan university of science and technology

<120> establishment method and application of anti-HIV (human immunodeficiency virus) drug evaluation animal model

<130> CP201221

<141> 2022-05-17

<160> 17

<170> SIPOSequenceListing 1.0

<210> 1

<211> 634

<212> DNA

<213> Unknown (Unknown)

<400> 1

tggaagggct aatttggtcc caaaaaagac aagagatcct tgatctgtgg atctaccaca 60

cacaaggcta cttccctgat tggcagaact acacaccagg gccagggatc agatatccac 120

tgacctttgg atggtgcttc aagttagtac cagttgaacc agagcaagta gaagaggcca 180

atgaaggaga gaacaacagc ttgttacacc ctatgagcca gcatgggatg gaggacccgg 240

agggagaagt attagtgtgg aagtttgaca gcctcctagc atttcgtcac atggcccgag 300

agctgcatcc ggagtactac aaagactgct gacatcgagc tttctacaag ggactttccg 360

ctggggactt tccagggagg tgtggcctgg gcgggactgg ggagtggcga gccctcagat 420

gctacatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480

gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540

tgagtgctca aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600

agaccctttt agtcagtgtg gaaaatctct agca 634

<210> 2

<211> 126

<212> DNA

<213> Unknown (Unknown)

<400> 2

atctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga 60

ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag 120

agcgtc 126

<210> 3

<211> 4307

<212> DNA

<213> Unknown (Unknown)

<400> 3

atgggtgcga gagcgtcggt attaagcggg ggagaattag ataaatggga aaaaattcgg 60

ttaaggccag ggggaaagaa acaatataaa ctaaaacata tagtatgggc aagcagggag 120

ctagaacgat tcgcagttaa tcctggcctt ttagagacat cagaaggctg tagacaaata 180

ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat 240

acaatagcag tcctctattg tgtgcatcaa aggatagatg taaaagacac caaggaagcc 300

ttagataaga tagaggaaga gcaaaacaaa agtaagaaaa aggcacagca agcagcagct 360

gacacaggaa acaacagcca ggtcagccaa aattacccta tagtgcagaa cctccagggg 420

caaatggtac atcaggccat atcacctaga actttaaatg catgggtaaa agtagtagaa 480

gagaaggctt tcagcccaga agtaataccc atgttttcag cattatcaga aggagccacc 540

ccacaagatt taaataccat gctaaacaca gtggggggac atcaagcagc catgcaaatg 600

ttaaaagaga ccatcaatga ggaagctgca gaatgggata gattgcatcc agtgcatgca 660

gggcctattg caccaggcca gatgagagaa ccaaggggaa gtgacatagc aggaactact 720

agtacccttc aggaacaaat aggatggatg acacataatc cacctatccc agtaggagaa 780

atctataaaa gatggataat cctgggatta aataaaatag taagaatgta tagccctacc 840

agcattctgg acataagaca aggaccaaag gaacccttta gagactatgt agaccgattc 900

tataaaactc taagagccga gcaagcttca caagaggtaa aaaattggat gacagaaacc 960

ttgttggtcc aaaatgcgaa cccagattgt aagactattt taaaagcatt gggaccagga 1020

gcgacactag aagaaatgat gacagcatgt cagggagtgg ggggacccgg ccataaagca 1080

agagttttgg ctgaagcaat gagccaagta acaaatccag ctaccataat gatacagaaa 1140

ggcaatttta ggaaccaaag aaagactgtt aagtgtttca attgtggcaa agaagggcac 1200

atagccaaaa attgcagggc ccctaggaaa aagggctgtt ggaaatgtgg aaaggaagga 1260

caccaaatga aagattgtac tgagagacag gctaattttt tagggaagat ctggccttcc 1320

cacaagggaa ggccagggaa ttttcttcag agcagaccag agccaacagc cccaccagaa 1380

gagagcttca ggtttgggga agagacaaca actccctctc agaagcagga gccgatagac 1440

aaggaactgt atcctttagc ttccctcaga tcactctttg gcagcgaccc ctcgtcacaa 1500

taaagatagg ggggcaatta aaggaagctc tattagatac aggagcagat gatacagtat 1560

tagaagaaat gaatttgcca ggaagatgga aaccaaaaat gataggggga attggaggtt 1620

ttatcaaagt aagacagtat gatcagatac tcatagaaat ctgcggacat aaagctatag 1680

gtacagtatt agtaggacct acacctgtca acataattgg aagaaatctg ttgactcaga 1740

ttggctgcac tttaaatttt cccattagtc ctattgagac tgtaccagta aaattaaagc 1800

caggaatgga tggcccaaaa gttaaacaat ggccattgac agaagaaaaa ataaaagcat 1860

tagtagaaat ttgtacagaa atggaaaagg aaggaaaaat ttcaaaaatt gggcctgaaa 1920

atccatacaa tactccagta tttgccataa agaaaaaaga cagtactaaa tggagaaaat 1980

tagtagattt cagagaactt aataagagaa ctcaagattt ctgggaagtt caattaggaa 2040

taccacatcc tgcagggtta aaacagaaaa aatcagtaac agtactggat gtgggcgatg 2100

catatttttc agttccctta gataaagact tcaggaagta tactgcattt accataccta 2160

gtataaacaa tgagacacca gggattagat atcagtacaa tgtgcttcca cagggatgga 2220

aaggatcacc agcaatattc cagtgtagca tgacaaaaat cttagagcct tttagaaaac 2280

aaaatccaga catagtcatc tatcaataca tggatgattt gtatgtagga tctgacttag 2340

aaatagggca gcatagaaca aaaatagagg aactgagaca acatctgttg aggtggggat 2400

ttaccacacc agacaaaaaa catcagaaag aacctccatt cctttggatg ggttatgaac 2460

tccatcctga taaatggaca gtacagccta tagtgctgcc agaaaaggac agctggactg 2520

tcaatgacat acagaaatta gtgggaaaat tgaattgggc aagtcagatt tatgcaggga 2580

ttaaagtaag gcaattatgt aaacttctta ggggaaccaa agcactaaca gaagtagtac 2640

cactaacaga agaagcagag ctagaactgg cagaaaacag ggagattcta aaagaaccgg 2700

tacatggagt gtattatgac ccatcaaaag acttaatagc agaaatacag aagcaggggc 2760

aaggccaatg gacatatcaa atttatcaag agccatttaa aaatctgaaa acaggaaagt 2820

atgcaagaat gaagggtgcc cacactaatg atgtgaaaca attaacagag gcagtacaaa 2880

aaatagccac agaaagcata gtaatatggg gaaagactcc taaatttaaa ttacccatac 2940

aaaaggaaac atgggaagca tggtggacag agtattggca agccacctgg attcctgagt 3000

gggagtttgt caatacccct cccttagtga agttatggta ccagttagag aaagaaccca 3060

taataggagc agaaactttc tatgtagatg gggcagccaa tagggaaact aaattaggaa 3120

aagcaggata tgtaactgac agaggaagac aaaaagttgt ccccctaacg gacacaacaa 3180

atcagaagac tgagttacaa gcaattcatc tagctttgca ggattcggga ttagaagtaa 3240

acatagtgac agactcacaa tatgcattgg gaatcattca agcacaacca gataagagtg 3300

aatcagagtt agtcagtcaa ataatagagc agttaataaa aaaggaaaaa gtctacctgg 3360

catgggtacc agcacacaaa ggaattggag gaaatgaaca agtagataaa ttggtcagtg 3420

ctggaatcag gaaagtacta tttttagatg gaatagataa ggcccaagaa gaacatgaga 3480

aatatcacag taattggaga gcaatggcta gtgattttaa cctaccacct gtagtagcaa 3540

aagaaatagt agccagctgt gataaatgtc agctaaaagg ggaagccatg catggacaag 3600

tagactgtag cccaggaata tggcagctag attgtacaca tttagaagga aaagttatct 3660

tggtagcagt tcatgtagcc agtggatata tagaagcaga agtaattcca gcagagacag 3720

ggcaagaaac agcatacttc ctcttaaaat tagcaggaag atggccagta aaaacagtac 3780

atacagacaa tggcagcaat ttcaccagta ctacagttaa ggccgcctgt tggtgggcgg 3840

ggatcaagca ggaatttggc attccctaca atccccaaag tcaaggagta atagaatcta 3900

tgaataaaga attaaagaaa attataggac aggtaagaga tcaggctgaa catcttaaga 3960

cagcagtaca aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt 4020

acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac 4080

aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag 4140

tttggaaagg accagcaaag ctcctctgga aaggtgaagg ggcagtagta atacaagata 4200

atagtgacat aaaagtagtg ccaagaagaa aagcaaagat catcagggat tatggaaaac 4260

agatggcagg tgatgattgt gtggcaagta gacaggatga ggattaa 4307

<210> 4

<211> 809

<212> DNA

<213> Unknown (Unknown)

<400> 4

atggaaaaca gatggcaggt gatgattgtg tggcaagtag acaggatgag gattaacaca 60

tggaaaagat tagtaaaaca ccatatgtat atttcaagga aagctaagga ctggttttat 120

agacatcact atgaaagtac taatccaaaa ataagttcag aagtacacat cccactaggg 180

gatgctaaat tagtaataac aacatattgg ggtctgcata caggagaaag agactggcat 240

ttgggtcagg gagtctccat agaatggagg aaaaagagat atagcacaca agtagaccct 300

gacctagcag accaactaat tcatctgcac tattttgatt gtttttcaga atctgctata 360

agaaatacca tattaggacg tatagttagt cctaggtgtg aatatcaagc aggacataac 420

aaggtaggat ctctacagta cttggcacta gcagcattaa taaaaccaaa acagataaag 480

ccacctttgc ctagtgttag gaaactgaca gaggacagat ggaacaagcc ccagaagacc 540

aagggccaca gagggagcca tacaatgaat ggacactaga gcttttagag gaacttaaga 600

gtgaagctgt tagacatttt cctaggatat ggctccataa cttaggacaa catatctatg 660

aaacttacgg ggatacttgg gcaggagtgg aagccataat aagaattctg caacaactgc 720

tgtttatcca tttcagaatt gggtgtcgac atagcagaat aggcgttact cgacagagga 780

gagcaagaaa tggagccagt agatcctag 809

<210> 5

<211> 477

<212> DNA

<213> Unknown (Unknown)

<400> 5

atggagccag tagatcctag actagagccc tggaagcatc caggaagtca gcctaaaact 60

gcttgtacca attgctattg taaaaagtgt tgctttcatt gccaagtttg tttcatgaca 120

aaagccttag gcatctccta tggcaggaag aagcggagac agcgacgaag agctcatcag 180

aacagtcaga ctcatcaagc ttctctatca aagcagtaag tagtacatgt aatgcaacct 240

ataatagtag caatagtagc attagtagta gcaataataa tagcaatagt tgtgtggtcc 300

atagtaatca tagaatatag gaaaatatta agacaaagaa aaatagacag gttaattgat 360

agactaatag aaagagcaga agacagtggc aatgagagtg aaggagaagt atcagcactt 420

gtggagatgg gggtggaaat ggggcaccat gctccttggg atattgatga tctgtag 477

<210> 6

<211> 2565

<212> DNA

<213> Unknown (Unknown)

<400> 6

atgagagtga aggagaagta tcagcacttg tggagatggg ggtggaaatg gggcaccatg 60

ctccttggga tattgatgat ctgtagtgct acagaaaaat tgtgggtcac agtctattat 120

ggggtacctg tgtggaagga agcaaccacc actctatttt gtgcatcaga tgctaaagca 180

tatgatacag aggtacataa tgtttgggcc acacatgcct gtgtacccac agaccccaac 240

ccacaagaag tagtattggt aaatgtgaca gaaaatttta acatgtggaa aaatgacatg 300

gtagaacaga tgcatgagga tataatcagt ttatgggatc aaagcctaaa gccatgtgta 360

aaattaaccc cactctgtgt tagtttaaag tgcactgatt tgaagaatga tactaatacc 420

aatagtagta gcgggagaat gataatggag aaaggagaga taaaaaactg ctctttcaat 480

atcagcacaa gcataagaga taaggtgcag aaagaatatg cattctttta taaacttgat 540

atagtaccaa tagataatac cagctatagg ttgataagtt gtaacacctc agtcattaca 600

caggcctgtc caaaggtatc ctttgagcca attcccatac attattgtgc cccggctggt 660

tttgcgattc taaaatgtaa taataagacg ttcaatggaa caggaccatg tacaaatgtc 720

agcacagtac aatgtacaca tggaatcagg ccagtagtat caactcaact gctgttaaat 780

ggcagtctag cagaagaaga tgtagtaatt agatctgcca atttcacaga caatgctaaa 840

accataatag tacagctgaa cacatctgta gaaattaatt gtacaagacc caacaacaat 900

acaagaaaaa gtatccgtat ccagagggga ccagggagag catttgttac aataggaaaa 960

ataggaaata tgagacaagc acattgtaac attagtagag caaaatggaa tgccacttta 1020

aaacagatag ctagcaaatt aagagaacaa tttggaaata ataaaacaat aatctttaag 1080

caatcctcag gaggggaccc agaaattgta acgcacagtt ttaattgtgg aggggaattt 1140

ttctactgta attcaacaca actgtttaat agtacttggt ttaatagtac ttggagtact 1200

gaagggtcaa ataacactga aggaagtgac acaatcacac tcccatgcag aataaaacaa 1260

tttataaaca tgtggcagga agtaggaaaa gcaatgtatg cccctcccat cagtggacaa 1320

attagatgtt catcaaatat tactgggctg ctattaacaa gagatggtgg taataacaac 1380

aatgggtccg agatcttcag acctggagga ggcgatatga gggacaattg gagaagtgaa 1440

ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag 1500

agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc 1560

ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga 1620

caattattgt ctgatatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa 1680

cagcatctgt tgcaactcac agtctggggc atcaaacagc tccaggcaag aatcctggct 1740

gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc 1800

atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt 1860

tggaataaca tgacctggat ggagtgggac agagaaatta acaattacac aagcttaata 1920

cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa 1980

ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata 2040

aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt 2100

tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca 2160

atcccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga 2220

gacagatcca ttcgattagt gaacggatcc ttagcactta tctgggacga tctgcggagc 2280

ctgtgcctct tcagctacca ccgcttgaga gacttactct tgattgtaac gaggattgtg 2340

gaacttctgg gacgcagggg gtgggaagcc ctcaaatatt ggtggaatct cctacagtat 2400

tggagtcagg aactaaagaa tagtgctgtt aacttgctca atgccacagc catagcagta 2460

gctgagggga cagatagggt tatagaagta ttacaagcag cttatagagc tattcgccac 2520

atacctagaa gaataagaca gggcttggaa aggattttgc tataa 2565

<210> 7

<211> 516

<212> DNA

<213> Unknown (Unknown)

<400> 7

atggtcttca cactcgaaga tttcgttggg gactggcgac agacagccgg ctacaacctg 60

gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg ggtgtccgta 120

actccgatcc aaaggattgt cctgagcggt gaaaatgggc tgaagatcga catccatgtc 180

atcatcccgt atgaaggtct gagcggcgac caaatgggcc agatcgaaaa aatttttaag 240

gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgcactatgg cacactggta 300

atcgacgggg ttacgccgaa catgatcgac tatttcggac ggccgtatga aggcatcgcc 360

gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa caaaattatc 420

gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat caacggagtg 480

accggctggc ggctgtgcga acgcattctg gcgtaa 516

<210> 8

<211> 581

<212> DNA

<213> Unknown (Unknown)

<400> 8

aattccgccc ctctccctcc ccccccccta acgttactgg ccgaagccgc ttggaataag 60

gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga 120

gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg 180

ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt 240

gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca 300

ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc 360

agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat 420

tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc 480

ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga 540

accacgggga cgtggttttc ctttgaaaaa cacgatgata a 581

<210> 9

<211> 634

<212> DNA

<213> Unknown (Unknown)

<400> 9

tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 60

cacaaggcta cttccctgat tggcagaact acacaccagg gccaggggtc agatatccac 120

tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 180

ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatggaatg gatgaccctg 240

agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 300

agctgcatcc ggagtacttc aagaactgct gacatcgagc ttgctacaag ggactttccg 360

ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420

gctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480

gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540

tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600

agaccctttt agtcagtgtg gaaaatctct agca 634

<210> 10

<211> 42

<212> DNA

<213> Unknown (Unknown)

<400> 10

tggaaaggat tttgctggat cctggtcttc acactcgaag at 42

<210> 11

<211> 43

<212> DNA

<213> Unknown (Unknown)

<400> 11

cggaattatc actagtggcg gccgcttacg ccagaatgcg ttc 43

<210> 12

<211> 21

<212> DNA

<213> Unknown (Unknown)

<400> 12

ggaccaggag cgacactaga a 21

<210> 13

<211> 23

<212> DNA

<213> Unknown (Unknown)

<400> 13

cagccaaaac tcttgcttta tgg 23

<210> 14

<211> 19

<212> DNA

<213> Unknown (Unknown)

<400> 14

gtgctaagca gttggtggt 19

<210> 15

<211> 21

<212> DNA

<213> Unknown (Unknown)

<400> 15

acagcctcaa gatcatcagc a 21

<210> 16

<211> 21

<212> DNA

<213> Unknown (Unknown)

<400> 16

atgagtcctt ccacgatacc a 21

<210> 17

<211> 25

<212> DNA

<213> Unknown (Unknown)

<400> 17

gtgctaagca gttggtggtg cagga 25

Claims (7)

1. A method for establishing an anti-HIV virus drug evaluation animal model, which takes an NPG mouse as a construction basis, comprises the following steps:

(1) hCD45 detection in mice by tail vein injection of human PBMC and/or CBMC cells from umbilical cord blood ⁺ Cell ratio when hCD45 ⁺ The cell proportion reaches more than 30 percent, and the humanized mouse model is judged to be successfully constructed;

(2) aiming at a mouse successfully constructed by a humanized mouse model, injecting HIV-Nanoluc virus liquid into the abdominal cavity for infection, imaging and detecting the proliferation condition of the virus in vivo every week after infection, and when the ROI value of a fluorescence signal reaches 10 ⁶ ～10 ⁷ The phototons/s are used for successfully modeling an animal model for evaluating the anti-HIV virus medicine;

the HIV-Nanoluc virus liquid has a nucleotide sequence shown in SEQ ID NO. 7.

2. The method according to claim 1, wherein said extraction of PBMC cells from peripheral blood of human origin comprises the steps of:

collecting peripheral blood of a healthy person, uniformly mixing the peripheral blood with heparin anticoagulant, performing centrifugal separation to collect upper plasma for later use, mixing the rest blood components with PBS preheated to room temperature in a volume ratio of 1:0.5-2, flatly paving the mixture on Ficoll lymphocyte separation liquid to form a clear separation interface, centrifuging, sucking a middle leucocyte layer into a clean centrifugal tube, adding PBS for cleaning, centrifuging, discarding supernatant, and cleaning for 1-3 times.

3. The method of claim 1, wherein the extraction of the human umbilical cord blood CBMC cells comprises the following steps:

collecting blood of umbilical cord of healthy human, mixing in heparin anticoagulant, centrifuging, collecting upper plasma, mixing the rest blood components with PBS preheated to room temperature at a volume ratio of 1:0.5-2, spreading on Ficoll lymphocyte separation liquid to form clear separation interface, centrifuging, sucking middle white membrane layer into a clean centrifuge tube, adding PBS for cleaning, centrifuging, discarding supernatant, and cleaning for 1-3 times.

4. The method of claim 1, wherein the caudal vein is injected with 4-6 x 10 human PBMC or CBMC cells ⁶ cell/cell.

5. The method of claim 1, wherein the HIV-Nanoluc virus solution is injected in an amount of 5-10 μ g/vial.

6. Use of an animal model established by the method of any one of claims 1 to 5 for screening for anti-HIV drugs.

7. The use according to claim 6, wherein the anti-HIV drug is VRC01-CAR-CD3 ⁺ T cell preparation, the VRC01-CAR-CD3 ⁺ The T cell preparation VRC01-CAR lentivirus transduced CD3 positive cells to obtain, the VRC01-CAR lentivirus consisted of signal peptide SP1, anti-HIV gp120 antigen specific single chain antibody VRC01, tag signal Strep tag II, linker peptide, CD8 hinge region, CD28 transmembrane region, 4-1BB co-stimulatory domain and CD3 zeta intracellular signaling domain.

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